The main objective of this proposal is to study the first specific enzyme in the cholesterol biosynthetic pathway, squalene synthase, in order to understand the mechanisms of its regulation. This objective is now obtainable since in the first three years of the project the investigators have obtained the cDNA clones of both the rat hepatic and the human hepatic enzymes as well as genomic clones of the 5' flanking sequences of the human gene. Based on work completed in the current grant period we now apply for a 5-year renewal in which the investigators propose to investigate the underlying mechanisms involved in the sterol-mediated transcriptional regulation of this gene. The investigators have identified a 69 bp promoter sequence which is responsible for this regulation and propose to investigate and elucidate the structure of the cis-acting elements which are involved in this regulation. It is further proposed to investigate the cellular trans-acting protein factors that are involved in the regulation of this gene and to compare them, and the regulatory mechanism to the regulation of other highly regulated enzymes in the cholesterol biosynthetic pathway. This will be achieved by the isolation and cloning of these protein factors and a detailed study of their interaction with the gene encoding squalene synthase. Based on results obtained in the current grant period the investigators propose to investigate the significance of the existence of different size-classes isoforms of squalene synthase mRNA in the regulation of this hepatic enzyme activity under varying cholesterogenic conditions. Finally, the investigators propose to verify the regulation of the human squalene synthase gene by its expression in transgenic mice. We also propose to study the implication of over production of this enzyme, in transgenic mice, on the overall metabolism of sterol and non sterol isoprenoids. The successful completion of these studies outlined in the grant application should enhance the understanding of the molecular basis of hepatic cholesterol production.